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From 2010.igem.org

Contents 1 Mimmi 1.1 yCCS and SOD 1.1.1 redoing the Site-Directed Mutagenesis 1.2 MITF 1.2.1 Amplifying 1.3 Gels 1.3.1 MITF 1.3.2 yCCS and RFP 1.3.3 yCCS and SOD 2 Nina 2.1 Mini prep of Tyrosinase 2.2 Measuring concentration of protein A (ZZ domain) and IgG protease in bank vector C 2.3 Ligation of protein A (ZZ domain) with bank vector C 2.4 Transformation of protein A (ZZ domain) in bank vector C 3 Andreas 3.1 Cloning of IgG protease 3.1.1 Colony PCR 3.1.2 Gel verification 3.2 New IgG protease cloning 3.2.1 Digestion 3.2.2 Ligation 3.2.3 Transformation

Mimmi

yCCS and SOD

redoing the Site-Directed Mutagenesis

Mix	(µl)	X2 X2		yCCS A		SOD A		conditions
sH2O	40	80		yCCS_mut_F		SOD_mut_F		time	°C
dNTPs	1	2		yCCS_mut_R		SOD_mut_R		30s	95
F primer	1	2		pSB1C3		pSB1C3		30s	95	)
R primer	1	2		~3.5kb		~3.2kb		30s	55	> 22 cycles
DNA	1	2X1						4m	68	)
Pfu buffer	5	10						oo	10
Pfu turbo	1	2
tot	50

SOD Primers
SOD_mut_F 262.55µl H2O --> 100µM
SOD_mut_R 372.64µl H2O --> 100µM
SOD_mut_F 3µl + 24.15µl H2O --> 125ng/µl
SOD_mut_R 3µl + 24.42µl H2O --> 125ng/µl

MITF

Amplifying

Mix phusion	(µl)	/4		Mix Pfu turbo	(µl)	/4			Primers
sH2O	67			sH2O	77				MITF_F
F primer	5			F primer	5				MITF_R
R primer	5			R primer	5
dNTP	2			dNTP	2			conditions
5X buffer	20			10X buffer	10			time	°C
Phusion pol.	1			Pfu pol.	1			2m	98
tot	100	25		tot	100	25		30s	98	)
								30s	40-55	> 5 cycles
								1m30s	72	)
								30s	98	)
								30s	65	> 25 cycles
								1m30s	72	)
								10m	72
								oo	10

Gels

MITF

verification

• Nothing...!

yCCS and RFP

verification

• See Andreas notes

yCCS and SOD

site-directed

well	sample
1	ladder
2	yCCS
3	blank
4	plasmid control
5	ladder
6	SOD
7	blank
8	plasmid control

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Nina

Mini prep of Tyrosinase

I performed a mini prep on the six samples of falcon tubes containing inoculated tyrosinase in its original vector that have been site directed mutagenesis. The colonies are: #2, 3, 4, 5, 6, ,7 & 8. The method was according to the procedure described under Protocols.

Spectophotometer:

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Measuring concentration of protein A (ZZ domain) and IgG protease in bank vector C

I measured the complately digested protein A ZZ domain (PCR product) and the bank vector C that before the digestion contained the IgG protease gene.

Spectophotometer:

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Ligation of protein A (ZZ domain) with bank vector C

I ligated protein A ZZ domian into the digested bank vector C to be able to make fusion proteins with IgG protease and CPP but also to send in this part to iGEM hq since they want the genes to be sent in this vector.

The vector concentraion should preferably be 25 ng/ul when performing ligation with a gene.

I used:

• bank vector C 0.5 ul
• protein A gene 3 ul
• quick ligase 1 ul
• quicke ligase buffer 2X 3 ul

Incubated in RT 15 min

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Transformation of protein A (ZZ domain) in bank vector C

I transformed the ligated protein A (ZZ domain) in bank vector C in 100 ul Top 10 cells.

The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 50 ul of sample on a chloramphenicol plate.

Andreas

Cloning of IgG protease

Continued from 9/8 ON plates

Colony PCR

Four clones (A, B, C, D) were picked for colony PCR. Positive control: pSB1C3.BBa_J04450 plasmid. Procedures according to protocol; 1:40 elongation time.

Gel verification

1 % agarose, 90 V, 40 min

Results: Weak and very irregularly sized bands. Probably something wrong with the ligation mixture. New IgGp digestion will be made.

New IgG protease cloning

Digestion

Used pSB1A3.IgGp at a conc. of 139 ng/μl.

10X FD buffer	2 μl
dH2O	3 μl
2 μg DNA	14 μl
FD SpeI	0.5 μl
FD EcoRI	0.5 μl
	20 μl

Incubation: 37 °C, 30 min

Ligation

Used pre-digested (EcoRI & SpeI) pSB1C3 vector from 3/8.

pSB1C3 vector	2.5 μl
IgGp insert	12.5 μl †
5X rapid ligation buffer	4 μl
T4 DNA ligase	1 μl

† Since I used the ligation protocol from 3/8, I mistakenly calculated that 12.5:2.5 would give a 5:1 insert:vector ratio. However, due to the new digestion volume, this actually led to a 12.5:1 ratio.

Transformation

Transformation into 100 μl comptetent Top10.

1. Quick-transformation
   1. 3 μl ligation mix. Incubation 5 min on ice.
   2. Heat-shock 30 sec in 42 °C. Cells cooled on ice.
   3. 100 μl plated onto Cm 25 plate.
2. Standard transformation
   • 3 μl ligation mix.
   • Standard protocol transformation procedures.

Plates incubated ON in 37 °C.